Dec. 20, 2007 A team of Vanderbilt researchers have demonstrated for the first time that a new type of gene therapy, called RNA interference, can heal a genetic disorder in a live animal.
The study shows that RNA interference can "rescue" a strain of mouse that has been genetically engineered to express a defective human hormone that interferes with normal growth. When the gene that produces the defective human growth hormone is inserted into the mouse's genome, it also stunts the mouse's growth. But when a small snippet of RNA that interferes with the hormone's production is also added, the mouse is restored to normal.
"It has been very satisfying to figure out the underlying cause of this genetic disorder and then identify a way to prevent it," says John Phillips, the David T. Karzon Professor of Pediatrics at the Vanderbilt University Medical Center, who has been studying human growth deficiency disorders since 1978. He collaborated on the research with graduate students Nikki Shariat and Robin Ryther, who are directed by Professor of Biological Sciences James G. Patton.
Growth hormone deficiency has been estimated to occur in between one in 4,000 to 10,000 children. It has a number of different causes, but one that is genetically inherited is called Isolated Growth Hormone Deficiency type II, and this is the subject of the study.
Children with IGHD-II appear fairly normal at birth but do not gain weight or grow as fast as they should, and their bones do not mature properly. The current treatment consists of daily injections of growth hormone for years until the patients reach their adult height. Not only is this treatment extremely expensive, it also fails to correct the underlying source of the problem: deterioration and death of cells in the pituitary gland that produce growth hormone. As a result, this single hormone deficiency can develop into multi-hormonal deficiency over time.
IGHD-II is what geneticists call a dominant negative disorder. It is caused by a defective form of human growth hormone that not only can't stimulate growth itself but also blocks the action of normal growth hormone. "It acts like Aesop's dog in the manger ... which has no use for the hay but keeps the cows from eating," says Phillips. Some other common dominant negative diseases include forms of colon cancer, deafness, muscular dystrophy, brittle bone disease, kidney disease and retinitis pigmentosa.
The blueprint for a protein like growth hormone is genetically encoded in a series of special segments called exons. The instructions in the exons are first copied onto a length of special RNA, called messenger-RNA. The messenger-RNA is moved to a structure in the cell called a ribosome, which links amino acids together in the order specified by the RNA sequence to create the protein.
Normal growth hormone is produced by a series of five exons. The defective hormone is the result of a splicing error: It is made by combining the segments coded by the first two exons and the last two exons, mistakenly skipping the third exon.
"A normal person has a very small amount of this defective hormone -- about 1 percent -- but people in families with IGHD-II produce 10 to 20 to 50 percent. And the more they make the slower they grow," says Patton.
In 2003, co-author Iain Robinson at the National Institute for Medical Research in London created a transgenic mouse with the human growth hormone gene that duplicated growth hormone deficiency. Although the altered mice still contained the mouse growth hormone genes, he found that high levels of the defective human growth hormone not only stunted their growth but actually killed the cells in the pituitary that produce growth hormone.
"This came as a real surprise: We never thought that a splicing error would lead to cell death," says Patton.
Meanwhile, progress in RNA interference research gave Patton and Phillips an idea for a way to correct this disorder.
In the last 15 years, scientists have realized that short pieces of double-stranded RNA, called silencing-RNA, use a pathway that is normally used by cells to regulate genes. This has created an opportunity for developing highly targeted therapies for a number of genetic diseases including macular degeneration in the eye and to block viruses such as herpes and RSV respiratory viruses. "To the best of our knowledge, this is the first time it has been used to correct a dominant negative disorder in a living animal," says Patton.
The researchers realized that the messenger-RNA that produced the defective hormone had a unique signature created by skipping the third exon. This allowed the Patton lab to create a specific silencing-RNA, designed to bind uniquely with the defective messenger-RNA.
"You might call this the 'if you don't like the message, kill the messenger' approach," Phillips quips.
Having created the special silencing-RNA, the next problem was how to deliver it to the pituitary gland which, in the case of the mouse, is the size of a grain of uncooked rice and is located at the base of the brain. As a proof of concept, the researchers decided to create a second strain of mouse which carried the special silencing-RNA and mate them with the growth deficiency strain. Their offspring should have both the genetic defect that produces the defective growth hormone and the silencing-RNA that should inhibit its production, allowing the mouse growth hormone to act.
The experiment was successful. The offspring grew normally and showed no defects in their pituitaries.
Now the researchers are investigating ways to deliver their silencing-RNA to the pituitary gland that would be suitable for treating humans. The cells that produce growth hormone have special receptors that signal the cells to release their stocks of growth hormone. If they can figure out a way to attach the silencing-RNAs to a compound that binds to this receptor, they should be able to deliver them to the cells where they can interfere with the activity of the defective growth hormone.
This research was published online Nov. 15 by the journal Endocrinology.
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